The Melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and controls food intake and energy expenditure. In humans, mutations in the MC4R are responsible for up to 5% of early onset obesity. Consequently, MC4R has been a target of the major pharmaceutical companies for the development of MC4R agonists to treat obesity. However, clinical trials of potent MC4R agonists, all of which have been orthosteric in nature, failed due to unwanted side-effects. Our goal is to develop allosteric modulators of the MC4R that will efficiently treat obesity without causing dangerous side effects. Based on the current knowledge on allosteric modulators, we hypothesize that a molecule that would potentiate MC4R signaling in an allosteric fashion would not only be more specific and more potent, but also safer than a direct orthosteric agonist. Indeed, allosteric modulators are less likely to cause side effects since they do not directly activate the target receptor but potentiate its response to endogenous agonist, in this case 1-MSH, in a more physiological temporo-spatial pattern. In addition, due to the lower conservation of allosteric sites, allosteric modulators are usually more specific than direct agonists. In order to identify such modulators of the MC4R, we have run a high-throughput screen (HTS) using the Vanderbilt compound library (160,000 compounds) and a system, based on the promega pGLO technology, that allows real time recording of cAMP responses in cells and the definitive identification of allosteric modulators. Hits identified by the screen were counterscreened for their activity at a similar Gs-coupled receptor (22-adrenergic receptor) in order to eliminate compounds that are not specific to the MC4R. 0.1% of the compounds from the library (166) were identified via this process as MC4R specific positive allosteric modulators. The first aim of this application will consist in characterizing these lead compounds using cell based pharmacological and biochemical methods. The most promising compounds will then be advanced to chemical optimization using technology-enabled synthesis to make and test large numbers of analogues of selected hits. The second aim will consist in testing in-vivo the best existing compounds as well as the most promising compounds that will arise from chemical optimization using the MC4R knockout mouse model of obesity. Indeed, WT, MC4R and MC4R-/- mice will be treated with vehicle or with the selected compounds while food intake, oxygen consumption and body weight will be monitored. Thus, the scientific goals of this training grant will be to develop small molecule drug-like compounds and use them to test in vivo the hypothesis that positive allosteric MC4R modulators may be useful for the treatment of syndromic, and possibly dietary obesity. The training goal will be to complement the candidate's existing skills in molecular pharmacology with extensive training in drug discovery technology, medicinal and chemical biology, small animal and transgenic/knockout husbandry, and small animal physiological and behavioral analysis. The candidate will have access to a leading laboratory in melanocortin receptors and obesity (mentor's laboratory), an academic drug discovery program with a proven record of success (Vanderbilt Institute for Chemical Biology), and leading NIH-funded source for mouse phenotyping (Vanderbilt Mouse Metabolic Phenotyping Center) to provide the training necessary to accomplish the program goals. PUBLIC HEALTH RELEVANCE: Haploin sufficiency of the melanocortin-4 receptor (MC4R) is responsible for up to 5% of severe early onset obesity. The development of potent allosteric modulators of the MC4R, proposed in this application, would provide a therapeutic approach to the treatment of this common Mendelian syndrome. The potential application of these compounds to common dietary obesity will also be examined.